The long-term goals of this project are to develop a high-resolution understanding of voltage-gated calcium channel (CaV) function and regulation. These molecular switches play pivotal roles in cardiac action potential propagation, neurotransmitter release, muscle contraction, calcium-dependent gene-transcription, and synaptic transmission. Calcium influx is a potent activator of intracellular signaling pathways but is toxic in excess. As a result, its entry into cells is tightly regulated. CaVs are major sources of activity-dependent calcium influx and possess a number of mechanisms that allow them to self-regulate. These mechanisms depend critically on interactions of the pore-forming subunit with cytoplasmic proteins that regulate channel activity. Our studies are aimed at understanding the molecular architecture that underlies CaV function and on developing novel reagents that can control channel function. We are investigating the hypothesis that two principal CaV inactivation mechanisms, calcium-dependent inactivation (CDI) and voltage-dependent inactivation (VDI) center on changes in the region of the selectivity filter. This is a paradigm-shifting view, based on our recent findings, that stands to align CaV inactivation mechanisms with a growing number of examples from other voltage-gated ion channel (VGIC) superfamily members. Due to the extraordinary challenges in studying mammalian membrane protein structure, part of our efforts focus on understanding basic structural mechanisms that are shared between CaVs and their ancestors, bacterial voltage gated sodium channels (BacNaVs). Production of multiprotein membrane proteins, such as CaVs, is a significant barrier to structural studies. To bridge this gap, we direct efforts to develop systems for production of full-length CaV complexes. In parallel, we investigate the how a novel class of reagents, anti-CaV? subunit nanobodies, interact with CaV? and modify channel function. Knowledge of such interactions will inform studies of how these novel, genetically-encodable reagents can be developed as versatile and selective agents to control CaV activity. Our studies integrate a multidisciplinary effort that includes biochemical, biophysical, X-ray crystallographic, cryo-electronmicroscopy, electrophysiological, and cell biology approaches. Because of their important role in human physiology, CaVs are the targets for drugs with great utility for the treatment of cardiac arrhythmias, hypertension, congestive heart failure, epilepsy, and chronic pain. Thus, understanding their structures and mechanisms of action in detail should greatly assist the development of valuable therapeutic agents for a wide range of human cardiac and neurological problems.